Juglans regia L.) varieties. The genetic control of these traits is still unknown in walnut, limiting the accuracy and rapidity of releasing new cultivars for commercial use. We studied the genetic architecture of five traits crucial for either marketing  or selection of individuals with specific phenology . By combining over 30 years of phenotypic data with genetic profiles generated using the latest Axiom™ J. regia 700K SNP array, we were able to identify and confirm major loci for all these traits. In particular, we revealed that a genomic region at the beginning of Chr1 controls both leafing and harvest date in walnut, consistent with the observed strong phenotypical correlation between these traits, and including candidate genes involved in plant development, leaf formation, and cell division. In addition, a large genomic region on Chr11 that includes genes with a central role in flowering control and shoot meristem growth underlies both lateral fruit-bearing and yield in walnut. We observed a more complex genetic architecture for pellicle color, strongly influenced by the environment (h2 = 0.43). We identified two marker-trait associations on Chr6 and 7 for pellicle color, where genes encoding a UDP-glycosyltransferase or involved in the response to oxidation were found. In conclusion, by combining classical quantitative trait loci (QTL) mapping and genome-wide association mapping, we deciphered, for the first time, the molecular pathways controlling walnut phenology, yield, lateral fruitfulness, and pellicle color. Our findings represent a further milestone in the transition from conventional to genome-assisted breeding in Persian walnut.Yield, nut quality, and ability to adapt to specific climate conditions, are all important factors to consider in the development and selection of new Persian walnut ( Juglans regia L.), native to the mountains of Central Asia, is widely cultivated in temperate regions extending from the Americas to Europe to Asia . In California, the first widely-planted cultivar to leaf out is “Payne,” with a bud-break in mid-March, while the latest is “Franquette,” which leafs out in the second half of April , was released and used to genotype a collection of 1,284 walnut accessions from the UCD-WIP  SNPs were used to test genotype-phenotype associations. SNP probes (71-mer) were mapped onto the new chromosome-level walnut reference genome (v2.0) (https://www.hardwoodgenomics.org/Genome-assembly/2539069) to obtain their chromosome locations. Specifically, a chromosome position was assigned to an SNP when its probe aligned uniquely on the genome with 98% identity and an E-value < 1e-20 for more than 95% of its length. The SNP dataset was then filtered for minor allele frequency (MAF > 5%) and missing rate (<20%). The genotypic dataset for this study can be found at https://hardwoodgenomics.org/Genome-assembly/2539069.The present research was based on 896 individuals of the UCD-WIP, which included several well-known walnut cultivars , advanced selections, and 34 full-sib families generated from controlled crosses in years 2004 to 2010. The largest family was “Chandler” x “Idaho” (CRxID) with 312 seedlings. Remaining families averaged 23 individuals each Table S1Historical phenotype data for leafing date (LefD), harvest date (HarD), lateral bearing (LTB), yield, and pellicle color were obtained from the UCD-WIP database. LefD is the time at which more than 50% of the terminal buds have begun to open. HarD is the time when 95% of the hulls have loosened from the shells. Both LefD and HarD were scored in Julian days. LTB is defined as the percentage of lateral buds flowering on elongated, 1-year-old shoots. Yield is based on visual evaluation with a score of 0 (no nut production) to 9 (highest yield). Pellicle color was scored using the official color chart of the Dried Fruit Association (DFA), where a score of one indicated extra-light, two—light, three—amber, and four—dark amber kernels. The averaged DFA score of 10 nuts per tree was then used as an individual record. Kinship relationships derived from the UCD-WIP pedigree  was usedYijklm is the m-th observation taken in the k-th year on the i-th tree of the j-th age class and located in the l-th block. In particular, ai is the additive genetic effect of the i-th tree on the trait, pei is the permanent environment contribution to the phenotype of the i-th tree, and eijklm is the residual term. The numerator relationship matrix was used to account for the covariance between additive genetic effects. A permanent environment effect was included in the model to account for the covariance between multiple observations from the same tree. For each trait, different models were fit, including both ai and pei, always as random effects, while age, year and block as random or fixed in a variety of combinations. The best model was then selected based on the Bayesian Information Criterion (BIC). Due to the categorical nature of yield, LTB, and DFA score, generalized linear mixed models were fit, relating the observed ordered categories to an underlying binomial density through a probit (yield) or logit (LTB and DFA score) link function. All models were fit and resolved using the REML estimation method implemented in ASReml v4 (h2) of each trait was then calculated as the ratio between the additive variance (σA) and the phenotypic variance (σP = σA + σPE + σe). Afterward, Pearson correlation value (R) between each pair of traits was investigated with R/GGally v1.4 . Also, Principal Component Analysis (PCA) based upon EBV values was performed using the built-in R function “prcomp.”where SReml v4 . The nar1 family “CRxID”   were removed. The linkage analysis was carried out using the R/ASMap package  using the Kosambi mapping function  . Markers package , which u package  to clustfunction . MapCharfunction  was usedp-value = 0.05) for each trait was determined with genome-wide permutation tests , and False Discovery Rate (FDR ≤ 0.05) were used to identify significant marker-trait associations.Five hundred eighty-four individuals, including all UCD-WIP families (except “CRxID”) and cultivars, were used to perform genome-wide association study (GWAS) for the five traits studied. GWAS was carried out by applying three different models implemented in R/GAPIT v3.0 : the Mulhttps://www.ncbi.nlm.nih.gov/genome/annotation_euk/Juglans_regia/100/) mapped onto the reference genome v2.0 to identify candidate genes for the studied phenotypes. We first inspected the LD landscape around the marker-trait associations identified in the same genomic regions by both QTL mapping and GWAS. By using the default method of confidence intervals implemented in Haploview v4.2 . LTB showed the highest h2 at 0.98, followed by LefD (0.88), yield (0.77) and HarD (0.68).Phenotypic records for all traits were collected by the UCD-WIP from 1988 to 2017 on trees ranging in age from one to 17 and distributed across 46 blocks. Both LefD and HarD were normally distributed , was appBased on individual breeding values estimated considering the entire walnut collection , the average HarD was 268.4 Julian days, with UC-06-005-8 the earliest tree (251 JD) and Scharsch Franquette the latest 290 JD; Table 1.R = 0.72; p-value < 0.001), suggesting that individuals with early leafing will also be early harvesting  with yield, suggesting higher yield would be expected in trees with early leafing and harvest dates. Also, the Pearson correlation analysis indicated that high-yielding individuals are generally lateral , and produce more extra-light kernels  spanned a total of 1,034.37 cM with 1,880 markers mapped into 16 LGs corresponding to the 16 walnut chromosomes Table 2.harD.1 explained 16.78% of the phenotypic variation , and also included the major QTL ltb.1 identified in the QTL mapping analysis. The trait with the second highest number of significant associations detected was LefD, with 14 SNPs found on Chr1, 4, 8, 10, 11, 12, 13, 16, and 17  and fell in two different LD blocks: the marker AX-170770379 formed a 5-kb LD block together with other two SNPs and was in strong LD with the most adjacent LD block, including 10 markers and spanning 25 kb  where no genes could be identified (The two SNPs on Chr1 associated with HarD were not in LD to each other r2 = 0.03 r2 = 0.03–0.35), except for the SNPs AX-171143099 and AX-170560823, which were moderately correlated (r2 = 0.53). The marker AX-170858858 formed a small LD block (< 1 kb) without any annotated candidate genes (r2 = 1) with four other SNPs, organized into only two haplotypes, GTCGG (frequency = 0.9) and TGTAC (frequency = 0.1). The other two most significant SNPs AX-171143099 and AX-170560823 fell in blocks of 43 and 13 kb, respectively. In the 43-kb block, four genes were identified . Six LD blocks of 75, 464, 49, 14, 300, and 151 kb were identified (5), 35 of which were also identified for LTB.Defining LD blocks was more complicated for LTB since the six most significantly associated SNPs covered a large genomic region of 12.5 Mb. Also, three SNPs  were in Hardy-Weinberg disequilibrium . Eight SNPs significantly associated with LTB fell in nine genes, encoding enzymes and transporters involved in carbohydrate metabolism processes, cellular signaling and ATP hydrolysis Table S4TMJ. regia 700K SNP array . Our study represents a further achievement in a long-term project of bringing molecular breeding in the UCD-WIP, which started with the release of the walnut reference genome  and moveNP array . Here weMalus domestica; Vitis vinifera L.; h2 estimates and those observed by h2 for LTB and yield, along with a more substantial proportion of lateral bearing individuals  and a higher average yield score. The value of h2 for HarD (0.68) was smaller than the one obtained by th) relative to the cv. Chandler (October 6th). We also estimate that 88% of the variability of LefD in our collection is heritable. A UCD-WIP goal is to release late leafing cultivars to avoid walnut blight, thus reducing the need for control measures   Table S22 ∼ 0) . Another2 ∼ 0) . HoweverBoth gene tagging strategies detected major QTLs for leafing and harvest date at the beginning of Chr1 4, S12During the spring, a complex signaling network mediated by plant hormones and transcription factors activates dormancy release, ending with bud break in response to warm temperatures . At thisAbout 1.7 Mb downstream from the candidate loci on Chr1 for LefD, we identified the protein TPX2-like gene as likely involved in HarD in walnut. The TPX2 protein is required for spindle assembly during mitosis . Walnut The strong selection exerted in favor of LTB and yield within the UCD-WIP during many years has created a large LD block on Chr11, where lack of recombination makes it difficult to select the best candidate gene for these two traits with precision.FLOWER LOCUS T-INTERACTING PROTEIN 1 (FTIP1), a key regulator of the transport of the FT protein from leaves to shoot apical meristems (SAM) for the initiation of flower development in response to day length and photoperiod , a DEAD-box helicase ATP-binding protein (OsABP) was upregulated in response to multiple abiotic stress treatments, including blue and red-light exposition  related to the use of EBVs as phenotypes for genetic mapping . The idehttps://hardwoodgenomics.org/Genome-assembly/2539069The genotypic dataset for this study can be found at AM coordinated the research, performed all the genetic analysis, and wrote the manuscript. GS assisted AM in analyzing and interpreting the data of pellicle color. CL provided the phenotypic data from the UCD-WIP database and his experience in walnut breeding to interpret the genetic results. HC contributed to fit the animal models for estimating breeding values and heritability. DN conceived and coordinated the research. All coauthors revised the manuscript.HC’s work is support by US Department of Agriculture, Agriculture and Food Research Initiative National Institute of Food and Agriculture Competitive Grant No. 2018-67015-27957.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despites numbers of case-control studies working on this area, the actual relationship of HIF-1α gene generic variant rs11549465 C>T imposing on cancer susceptibility remains unveiled. To get a better understanding of such relationship, this meta-analysis was carried out by incorporating all eligible case-control studies. Qualified articles were acquired from PubMed, CNKI, EMBASE, PMC, and Wanfang database update to April 2019. Odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were employed to estimate the relationship of interest. Heterogeneity tests, sensitivity analyses and publication bias assessments were also carried out to ensure the strength of our conclusion. A total of 46 articles with 49 studies including 12920 cases and 13363 controls were included. The results indicated that HIF-1α rs11549465 C>T was significantly related to the increased risk of overall cancer under four genetic models . Furthermore, enhanced cancer risk was detected after stratification by cancer type, ethnicity, the source of controls and HWE. These results suggest that HIF-1α rs11549465 C>T polymorphism may predispose to cancer susceptibility.HIF-1 Cancer ranks itself the leading causes of death around the world. In 2019, 1,762,450 new cancer cases and 606,880 cancer deaths are projected to occur in the United States. It has become a universal public health issue α plays a vital role in activating various genes that is significantly involved with cell adhesion, erythropoiesis, angiogenesis and glucose transportation in the process of cancer development and progress Hypoxia possesses a vital role in the maintenance of tumor microenvironments. Hypoxic tumor microenvironment triggers extensive cellular responses, such as angiogenesis, proliferation and invasion HIF-1α has been a research hot spot and numerous SNPs in HIF-1α were identified, whose polymorphism known as 1772 C>T , having been the most widely investigation polymorphism. rs11549465 C>T is a nonsynonymous SNP. Compared to the wild type, this polymorphic variant can tremendously enhance transcriptional activity in both normoxic and hypoxic environment in in-vitro studies HIF-1α rs11549465 C>T is linked to increased tumor microvessel density which makes contribution to the cancer progression. HIF-1α rs11549465 C>T polymorphism was previously investigated in various types of cancer. Nevertheless, the conclusions obtained from previous epidemiological studies are inconsistent and contradictory. Thus, the relationship between HIF-1α rs11549465 C>T polymorphism and cancer risk requires further exploration. Herein, we performed this more comprehensive meta-analysis on selected case-control studies in the aim of giving a more thorough demonstration of the association of HIF-1α rs11549465 C>T polymorphism with cancer risk.Mounting evidence provided that featuring a high tumor grade, HIF-1α is over-stated in numbers of human cancers, indicating that HIF-1α functions as an independent element of cancer prognosis hypoxia-inducible factor-1 or HIF-1α or rs11549465 or 1772 C>T; 2) SNPs or polymorphisms or polymorphism or variants; 3) cancer or carcinoma or neoplasm or tumor. To acquire all qualified publications, we also reviewed the references of the selected studies.We systematically searched EMBASE, PubMed, PMC, Wanfang and CNKI to retrieve relatively pertinent publications based on case-control studies . No language restriction is made for this analysis. The search terminology involved were as listed: 1) Impertinent and irrelevant studies were excluded on primary stage. Elimination criteria were: if 1) the study population was not mapped out; 2) it is not case-control study; 3) lack of information in allele frequency. Other than that, editorials, reviews and meta-analysis were ruled out. Only case-control studies with detailed number of different genotypes for estimating odds ratios (ORs) with 95% confidence intervals (CIs) were taken into the final analysis.Two authors (Hu-Nian Li and Ting He) were arranged to extract information of all the articles respectively. Items listed below were extracted from every single study: 1) authors name; 2) publication year; 3) ethnicity of the study subject; 4) cancer type; 5) allelic frequency; 6) quality score. Studies with scores ≤9 were of low quality, whereas those with scores >9 were of high quality HIF-1α rs11549465 C>T polymorphism and cancer risk by calculating ORs with the corresponding 95% CIs. Moreover, we conducted the stratification analysis by ethnicity, cancer type, source of control, and HWE in controls. We also used Chi square-base Q-test to gauge the presence of heterogeneity. The fixed-effect model was used to compute the pooled OR, given the studies were confirmed to be homogeneous (P>0.10 for the Q test). Or the random-effect model should be used instead. Sensitivity analysis was undertaken on the base of re-calculation of the ORs and 95% CIs by excluding each study individually. In order to detect the presence of publication bias, Begg's funnel plot and Egger's linear regression were adopted simultaneously. We also performed the trial sequential analysis (TSA) to avoid the random errors caused by repeated significance testing and dispersed data P value <0.05 as a baseline significant finding.We first performed Hardy-Weinberg equilibrium (HWE) for the controls utilizing the goodness-of-fit test. Homozygous model, heterozygous model, recessive model, dominant model, and allele model were employed to determine the relationship between Figure 1. We first collected 196 articles of the interest by a comprehensive search in the above-mentioned databases. After a basic check-up on articles relevance and abstracts conciseness, 156 articles were ruled out, which left us a total of 40 articles for full text assessment. To expand its sample size to ensure statistical representativeness, we identified another 6 articles from retrieve studies, quantity adding up to 46 articles in total Table .The study workflow was graphically displayed in Table 2 and Figure 2. The results concluded that the rs11549465 C>T polymorphism was significantly related to the increased risk of overall cancer under homozygous model , recessive model ; dominant model , and allele model . We failed to detect any distinguished relationship between rs11549465 C>T and renal cell carcinoma (RCC), endometrial cancer, colorectal cancer, lung cancer, breast cancer, hepatocellular cancer (HCC) under all the five genetic models. However, we observed that the rs11549465 C>T polymorphism could confer to increased risk in subgroups of prostate cancer , cervical cancer , oral cancer , pancreatic cancer  and other cancers .The quantitative results of the meta-analysis were displayed in When it comes to the stratification analysis by the ethnicity, significant increased risk was detected in Asians, Caucasians and mixed population. In terms of source of controls, either population-based controls or hospital-based controls were associated with the increase risk of cancer. Further subgroup analysis by HWE in controls revealed that no significant correlation was observed in subgroup of HWE≤0.05. As regard to the quality of publications, significant increased risk was detected in high-quality and low-quality publications.Q test (P<0.001) implied an existence of heterogeneity under all the genetic models. Thus, we adopted a random-effect model to produce ORs and 95% CIs. In addition, the sequential sensitivity analysis was performed to give an evaluation of the impact of a sole study on the pooled estimation. Given the attempt of omitting in each study incurred no statistical fluctuation of the pooled ORs, we have reason to believe that the meta-analysis's data is of great reliability .The Figure 4, no evidence of asymmetry was found. Egger's test's statistics also gives no evidence of publication bias among the studies.From the shape of the Begg's funnel plot shown in Figure 5).The TSA showed that the cumulative z-curve did not cross both the traditional threshold and the TSA threshold, yet the accumulated information was sufficient, indicating that no further evidence was needed to verify the conclusion  could induce proline-to-serine amino acid substitutions. However, the exact role of rs11549465 C>T polymorphism in cancer risk obtained from different studies remain inconclusive.The HIF-1α rs11549465 C>T and cancer risk. However, association analysis between rs11549465 C>T and RCC risk in panels of 20 cases and 44 non-neoplastic controls did not reveal allelic frequency differences. An investigation conducted by Konac et al. HIF-1α may contribute to risk of endometrial and cervical cancers. In a meta-analysis performed by Zhao et al. HIF-1α rs11549465 C>T polymorphism was reported to be related to increased cancer risk. In 2015, Li et al. HIF-1α rs11549465 C>T polymorphism predispose to higher overall cancer risk. To better illustrate the relationship of interest, we hereby conducted this updated meta-analysis by using all the qualified publications with a total of 12920 cases and 13363 controls. The results revealed that HIF-1α rs11549465 C>T polymorphism contributes to increased overall cancer risk. In a sense, this meta-analysis has succeeded in giving a clearer clue of the relationship between HIF-1α rs11549465 C>T polymorphism and cancer risk.In 2001, Clifford et al. In the current meta-analysis, we undertaken many measurements to increase the credibility of our conclusion. First and foremost, we included as many as qualified studies to expand the analyzed sample size, by incorporating studies not only pressed in English but also in Chinese. Second, we adopted the sensitivity analysis and the publication bias. However, several limitations could not be settled down. First, between-study heterogeneity exists, which might weaken the persuasiveness of the conclusion. Second, the relationship strength was only assessed by use of unadjusted estimates. Lacking original data, such as environment factor, adjustment analysis was absent. Third, most of the included studies were conducted among Asians and Caucasians. The lack of other ethnicities, such as Africans, compromised the generalization of the conclusion.HIF-1α rs11549465 C>T polymorphism was significantly related to an increase in cancer risk. Our work no doubt will encourage more dedication into further elucidation of the etiology of cancer predisposition. However, with limited sample size of subgroup analysis, we must admit that this analysis is imperfect and thus in the future more case-control studies should be conducted with a larger size of samples.In a word, our finding has come to a fruition that


Radium-223 is a first-in-class targeted alpha therapy to prolong overall survival (OS) in castration-resistant prostate cancer with bone metastases (mCRPC). The aim of the present analysis was to assess the long-term safety with radium-223 in Japanese patients with mCRPC.Patients with symptomatic mCRPC, ≥ 2 bone metastases and no known visceral metastases received up to 6 injections of radium-223 (55 kBq/kg), one every 4 weeks. Adverse events (AEs) considered to be related to radium-223 were reported until 3 years after the first injection. Pre-specified conditions, such as acute myelogenous leukemia, myelodysplastic syndrome, aplastic anemia, primary bone cancer, or other primary malignancies, were reported regardless of causality.Of the 49 patients enrolled in the study, 44 (89.8%) entered the survival follow-up period and 33 (67.3%) died. Throughout the entire study, there were no reports of second primary malignancy or other pre-specified conditions. Eight patients (16.3%) experienced post-treatment drug-related AEs, which were all hematological . No serious post-treatment drug-related AEs were reported. Updated median OS was 19.3 months .In Japanese patients with symptomatic mCRPC and bone metastases, radium-223 had a favorable long-term safety profile with no second primary malignancies reported. Taken together with median OS, which was comparable to that in the pivotal phase III ALSYMPCA study, these results support continued benefit from radium-223 in Japanese patients with mCRPC.The online version of this article (10.1007/s10147-018-01389-4) contains supplementary material, which is available to authorized users. Bone is one of the most common metastatic sites among patients with prostate cancer . In patip = 0.002) [p < 0.001 for both), and was well tolerated [Radium-223 dichloride (radium-223) is a first-in-class targeted alpha therapy that has been developed for the treatment of skeletal metastases , 8. Due = 0.002) . In ALSYolerated . In addiolerated .n = 49), intravenous radium-223 was associated with a mean change in total ALP from baseline to 12 weeks (primary endpoint) of − 19.3%  [In a phase II study of Japanese patients with symptomatic mCRPC and bone metastases ( − 10.7) . The ALP − 10.7) .The aim of the present analysis was to assess the long-term safety, as well as updated OS, associated with radium-223 in Japanese patients with symptomatic mCRPC and bone metastases enrolled in this phase II study using follow-up data of 3 years since the first administration.The study protocol was approved by each study center’s independent ethics committee or institutional review board, and the study was conducted in accordance with the ethical principles of the Declaration of Helsinki and the International Conference on Harmonization guideline E6: Good Clinical Practice. Written informed consent was obtained from all patients.The design of this multicenter, single-arm, open-label, phase II study (NCT01929655) has been previously described . BrieflyAll enrolled patients received intravenous radium-223 at a dose of 55 kBq/kg on day 1 of each 4-week cycle for a maximum of six cycles.The study consisted of a treatment period, defined as the time from the first injection to end of treatment , an active follow-up period (from EOT to 12 weeks thereafter), and a survival follow-up period (from 12 weeks after EOT to 3 years after the first injection). Efficacy data, as well as SSEs, were assessed at visits conducted on day 1 of each treatment cycle, at EOT, and once every 4 weeks during the active follow-up period. Concomitant or subsequent therapy started within 30 days after the last injection was recorded. During the survival follow-up period, patients were contacted every 6 months, either over the telephone or by visits, and survival was assessed.TEAEs  were reported regardless of causality. Investigators were required to report post-treatment AEs, which occurred more than 30 days after the last injection, only if they were considered drug-related (post-treatment drug-related AE). All instances of acute myeloid leukemia (AML), myelodysplastic syndrome, aplastic anemia, and bone cancer, as well as any other second primary malignancies, were considered serious AEs (SAEs) and were reported regardless of the time of occurrence or causality.Statistical analyses were performed using the software package SAS release 9.2. The Kaplan–Meier method was used to estimate median OS, while median survival follow-up time was estimated by the reverse Kaplan–Meier method . DescripOf the 49 patients with mCRPC who were administered radium-223, 45 91.8%) entered the active follow-up period, while 4 patients (8.2%) did not , while 17 and 10 patients experienced Grade 1–2 and Grade 3–4 drug-related AEs in treatment period. Of these three patients, one experienced clinical disease progression and discontinued radium-223 at the occurrence of the AE (decreased platelet count). The use of post-radium chemotherapy before worsening of the AE was not reported for any of these patients. Due to the fact that TEAEs and post-treatment drug-related AEs were collected differently, the data on the incidence of drug-related TEAEs are presented for reference only and include only the AEs which were also reported as post-treatment drug-related AEs. Voluntarily reported non-related AEs are summarized in Supplementary Table 2.Of the 49 patients in the safety analysis set , 8 patients 16.3%) experienced post-treatment drug-related AE; all of these events were hematologic toxicities . Two patients experienced fractures during the treatment period (6 and 47 days after the first injection of radium-223), and one during the active follow-up period . None of the fractures were considered serious or related to the study drug. All fractures were classified as Grade 2 AEs. None of the patients who had fractures had used bone-modifying agents, either before or concomitantly with study drugs.After a median follow-up time of 35.9 months, 32 death events had occurred. In addition, 1 patient with an unknown date of death was censored. The median OS was 19.3 months , Grade 3 AEs were relatively infrequent (2–4%) in the present analysis. In addition, all patients who reported these events had already experienced the same hematologic AEs during the treatment period. This indicates that late-onset new hematologic AEs with radium-223 are rare.Because radiotherapy, for example EBRT, increases the risk of secondary malignancies , radium-In another phase 3 study (ERA 223), in which radium-223 or placebo was initiated at the same time as abiraterone acetate plus prednisone/prednisolone in asymptomatic or mildly symptomatic patients with mCRPC and bone metastases , prelimiIn the primary analysis of this phase II study of Japanese patients with symptomatic mCRPC and bone metastases, decreased ALP levels at 12 weeks (primary endpoint) were observed following radium-223 therapy , consistIn the exploratory subgroup analyses, a trend for a longer OS was seen with several patient subgroups; including patients with higher albumin, lower neutrophil, and lower LDH levels, those without a prior history of docetaxel, patients with an EOD of 1/2, and patients with higher hemoglobin, lower ALP and lower PSA levels; however, it is important to note that these findings are not conclusive because they were selected by arbitrary cutoff . These trends were generally consistent with previously reported prognostic factors in patients with mCRPC –26. A trThe present analysis had a number of limitations. Firstly, the study had a single treatment arm with a relatively small sample size designed for the evaluation of ALP change; therefore, it was underpowered for other outcomes, including safety, OS, and the subgroup analyses. Furthermore, follow-up was limited to 3 years, which may not be long enough to fully assess the number of treatment-related malignancies. Additional studies with larger sample size are needed to confirm the long-term safety. At present, an international, prospective, observational, single-arm study , in which patients will be followed until 7 years after the last radium-223 dose, is ongoing.In conclusion, in this 3-year follow-up of Japanese patients with symptomatic mCRPC and bone metastases, no new safety concerns were identified and no second primary malignancies were reported, suggesting that radium-223 has a favorable long-term safety profile in Japanese patients. Median OS with radium-223 was at least as favorable as that observed in ALSYMPCA. When considered together with the decrease in ALP levels associated with radium-223 and reported in the primary analysis, it is likely that the efficacy findings of the present study are consistent with those of ALSYMPCA. The safety and efficacy results of the present study suggest that radium-223 provides long-term benefits in Japanese patients with mCRPC and bone metastases.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 41 KB)


In the present study, we explored the role of the interferon-inducible protein p202 in osteoblast differentiation of mouse bone marrow stromal cells (BMSCs). Both the mRNA and protein levels of p202 increased initially and decreased afterward in the course of BMSC osteogenesis. The intracellular distribution of this protein also changed in the differentiation process. p202 knockdown inhibited, while p202 overexpression enhanced, the osteoblast differentiation of BMSCs. This was identified by evaluation of expression of osteogenic markers, Alizarin Red S staining, and determination of alkaline phosphatase activity. Further study revealed that p202 disturbs the formation of Runx2/Ids complex and frees Runx2 to induce the differentiation process. The findings demonstrated that p202 plays a positive role in BMSC osteogenesis. As a regulator that coordinates calcium and phosphate homeostasis, bone plays various physiological roles in vertebrates. It is an endocrine and hematopoietic organ as well, by secreting bone-derived hormones and hemocytes, which may regulate glucose homeostasis, kidney function, and even food intake ,2. Bone The murine interferon-inducible p200 family is composed of more than ten homologous proteins including p202, p203, p204, p205, Aim2, Mndal et al. . These fIn the present study, we first examined the temporal and spatial expression of p202 during the osteoblast differentiation of mouse BMSCs. Next, effects of p202 knockdown or overexpression on BMSC osteogenesis were determined, with regard to the alterations in transcription of osteogenic genes, mineralization, and alkaline phosphatase (ALP) activity. In addition, the underlying mechanisms were elucidated via co-immunoprecipitation (Co-IP) assay and luciferase reporter gene assay.2 at 37°C. Four days later, the nonadherent cells were removed. The adherent cells were cultured and passaged as previously described [The present study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Animal Care and Use Committee of Shandong province, China. The study was approved by the Animal Research Ethical Committee of Shandong province, China. BMSCs were acquired by accumulating the bone marrow of femurs and tibiae from Kunming mice . Bone marrow cells suspended in DMEM were transferred into cell culture dish and cultured in an incubator with 5% COC−∆∆t method.Total cellular RNA was extracted using RNAiso plus reagent . RNA samples were reversely transcribed into cDNA using a reverse transcription kit (Takara). The mRNA levels of specific lineage markers were measured by qRT-PCR. The primer sequences for p202, GAPDH, Runx2, Col I (Collagen type I), and OCN  were listed in Total cell proteins, cytoplasmic, and nuclear fractions were collected respectively, according to the manufacturer’s instructions . Aliquots of protein from each sample were used to determine the expression patterns of p202 and Runx2, with GAPDH or Histone H3 as the internal control. The antibodies were from Abcam or Santa Cruz Biotechnology. Proteins were subjected to SDS-PAGE on a 12% polyacrylamide gel and transferred onto a PVDF membrane . After blocking with 5% nonfat milk in TBST (TBS containing 0.05% Tween-20), the membrane was incubated with the primary antibody and subsequently with the horseradish peroxidase-conjugated secondary antibody. The immunoreactive proteins were detected via chemiluminescence using an ECL kit . The images of Western blotting were quantified by Image-Quantity software .Knockdown of p202 gene was performed using RNA interference technique. The specific nucleotide sequences of siRNA-control (nonspecific RNA duplex) and siRNA-p202 (siRNA specific to mouse p202 gene) were listed in XhoI/HindIII fragments encoding the wild-type mouse p202 were cloned into pcDNA3.1(+), according to the manufacturer’s protocol. Briefly, the full length cDNA of p202 was amplified by a dynamic template PCR technique, using primers containing XhoI or HindIII restriction sites. The cDNA template and the pcDNA3.1(+) vector were separately cutted by XhoI and HindIII enzymes. The cutted cDNA fragments were ligated into the pcDNA3.1(+) vector  to form pcDNA-p202. The correctness of the construct was confirmed by sequencing. BMSCs were treated as described above and transfected with either vector alone (pcDNA) or the p202-containing vector (pcDNA-p202) (400 ng/ml) employing Lipofectamine 2000.To construct the recombinant p202 overexpression plasmid, the p-nitrophenol from p-nitrophenyl phosphate at 37°C using an ALP assay kit . Production of 1 mg p-nitrophenol at 37°C in 15 min was described as 1 unit (U). ALP activity was normalized to total protein determined using a BCA assay kit (Beyotime), which expressed as U/g protein.On day 4, treated cells were washed with PBS and lysed by two cycles of freezing and thaw. ALP activity in the cell layer was measured colorimetrically by monitoring the release of On day 8, ARS staining was performed to detect the mineralized nodules in treated cells, as previously described . Briefly. Treated cells were harvested and aliquots were incubated with the anti-p202 or anti-Runx2 antibodies (1 μg) at 4°C overnight. The cell extracts were removed after being incubated with protein A/G Plus Agarose beads. The proteins connected to the beads were released and examined by Western blotting with anti-p202, anti-Runx2, anti-Id1, anti-Id2, or anti-Id3 antibodies.Co-IP assay was employed to test the binding of p202 or Runx2 to other proteins functioning in osteogenic differentiationTo obtain luciferase reporter constructs, the appropriate DNA segments containing the promoter region of OCN were cloned into the pGL3-basic vector, which named as pGL3-OCNp. The primers with restriction enzyme sites were shown in t-test. P<0.05 was significant in statistical analysis.All experiments were conducted at least three times. Data were expressed as average ± SD. Origin software was used to assay the differences among samples, either by the one-way ANOVA or by two-sample Student’s The mRNA and protein levels of p202 during BMSC osteogenesis were determined by qRT-PCR and Western blotting respectively. As shown in The subcellular distribution of p202 was detected by immunoblotting using cytoplasmic and nuclear extracts at the indicated days . In the To test whether p202 is required for osteoblast differentiation, siRNA-p202 was used to silence the expression of p202. BMSCs were transfected with siRNA-p202, siRNA-control, or mock. Results in The transcription of genes typically induced during osteogenesis was down-regulated by siRNA-p202 C. For inWe also tested the impact of p202 overexpression on osteoblast differentiation. As shown in Runx2 is a critical transcription factor in osteogenesis . Id protIn order to further detect the role of Id proteins in the effect of p202 on osteogenesis, protein extracts from mock, siRNA-p202, pcDNA, or pcDNA-p202-treated cells were immunoprecipitated with anti-p202 antibody, and the complexes were detected with anti-p202 and anti-Id2 antibodies. Results indicated that p202 bound to Id2 in all the treated cells. SiRNA-p202 suppressed the p202 protein level significantly and also markedly reduced the interacted Id2 protein content C. HoweveResults in As shown in The interferon-inducible p200 family proteins are the gene products induced by interferons, which are important regulators in cell growth and differentiation . p202 isIn the first, the expression pattern and subcellular location of p202 during BMSC osteogenesis were determined. Both the mRNA and protein levels were induced significantly after the start of the differentiation process, with day 4 and 6 showing maximal levels respectively . This reTo fully assess the role of p202 in BMSC osteogenesis, silencing and overexpression of this protein were achieved using a specific small interfering RNA and a recombinant plasmid respectively. Results demonstrated that p202 knockdown inhibited, while its overexpression enhanced, the osteoblast differentiation of BMSCs  and 4. TFinally, the mechanism by which p202 activates BMSC osteogenesis was determined. Runx2 is a critical transcription factor in osteogenesis. Previous study has detected the association of p204 with Runx2 . However


N = 63 mother–infant dyads at study entry, n = 28 diagnosed with postpartum anxiety disorders according to the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV), n = 35 were healthy controls. Mothers were interviewed with the Structured Clinical Interview for DSM-IV Disorders at an average infant age of M = 4.1 months and filled out a questionnaire regarding emotional stress during pregnancy. Further, they were videotaped during the Face-to-Face-Still-Face paradigm (FFSF), a widely used mild socio-emotional stressor for infants. To determine infant stress-reactivity, infant salivary cortisol was collected before, immediately after and 20 min after the FFSF. Missing values were estimated by multiple imputations. At the age of M = 5.3 years, mother-child-interaction was re-assessed in a follow-up sample of n = 30 dyads via a free-play situation. Moreover, dimensional measures for anxiety were assessed. Mothers in the clinical group reported significantly higher stress scores than the control group. Infant stress reactivity in the early postpartum period and maternal anxiety symptoms at the 5-year follow-up assessment were significantly associated with dyadic interaction quality at pre-school age. Even though maternal stress during pregnancy did not directly predict mother–child interaction quality at pre-school age, it was significantly correlated with infant cortisol reactivity during postpartum period. Nevertheless, caution should be taken when interpreting the results considering the small sample size.There is growing evidence that even milder forms of maternal stress or anxiety during pregnancy affect the fetus causing possible long-term consequences for infant and child development. The mechanisms through which prenatal maternal stress may affect the unborn are not yet entirely clarified. Due to limited self-regulatory skills after birth, infants depend on sensitive behavior of their parents to regulate affective states and physiological arousal. Dyadic affect regulation has been linked to various developmental patterns up to adolescence and thereby represents a key element of early social relationships. Aim of the study was to evaluate possible long-term consequences of emotional stress during pregnancy and postpartum anxiety disorders, as well as infant postpartum cortisol reactivity on mother–child-interaction at pre-school age. The sample comprised of  A growing body of research indicates that maternal stress during pregnancy exerts strong influence on the development of the unborn . Recent To date, prenatal maternal stress is defined very broadly, including psychological distress such as anxiety or depressive symptoms and life events, e.g., trauma, loss, or natural disasters. In this study we focused on emotional stress during pregnancy. This was assessed retrospectively with a questionnaire in the early postpartum period, including items regarding maternal experience of anxiety, sadness, joy, stress, and general tension .Maternal anxiety disorders in the perinatal period are the most common psychiatric disorders with prevalence rates of 11 to17%  and are Even though it is difficult to disentangle the influence of prenatal and postnatal maternal stress on infant and child development, current studies try to differentiate between timing effects. Prenatally, maternal emotional stress seems to affect cognitive development. In a study of A growing body of studies find significant links between prenatal stress and/or anxiety and child development up to adolescence  is part of a larger longitudinal study . Women were only included in the control group if they had no current or antecedent axis I diagnoses according to DSM-IV.This sample , N = 64 mothers agreed upon selection of their infant’s saliva. n = 1 infant was excluded due to cortisone medication. None of the infants met the exclusion criteria of prematurity (born before the completion of the 36th week of gestation), small-for-gestational-age or congenital abnormalities. Thus, the final sample consisted of N = 63 mother–infant dyads at study entry. n = 28 of the mothers were diagnosed with at least one anxiety disorder  and n = 35 mothers had no clinical disorder (control group). A detailed report of total recruitment and case exclusions is demonstrated in At first assessment n = 20 mothers had more than one anxiety disorder: n = 14 mothers had a panic disorder with or without agoraphobia or an agoraphobia without history of panic disorder. n = 14 mothers were diagnosed with a generalized anxiety disorder. n = 13 mothers were diagnosed with an obsessive-compulsive disorder. n = 9 mothers had a social phobia. n = 9 women were diagnosed with a specific phobia. n = 1 woman had a post-traumatic stress disorder. n = 2 mothers had an anxiety disorder NOS. The vast majority of the clinical sample  was diagnosed with a prepartum onset of their anxiety disorder(s).The distribution of diagnoses in the clinical group was as follows: N = 32 mother–infant dyads could be reached . This high dropout rate was primary due to life changes in the recruited families (change of address and contact data) and changes of interests (rejection of or non-response to study invitation) within the assessment gap of 5 years. n = 6 still suffered from at least one current anxiety disorder and further n = 3 from at least one partially remitted anxiety disorder. Of these, n = 1 mother had four and n = 1 mother had three anxiety disorders. Overall, there was n = 1 mother with a current agoraphobia without history of panic disorder and n = 1 mother with a fully remitted panic disorder. n = 2 women were diagnosed with a current generalized anxiety disorder ; n = 6 women suffered from an obsessive-compulsive disorder ; n = 1 woman had a social phobia; n = 2 mothers were diagnosed with a specific phobia; and n = 1 woman was diagnosed with a partially remitted anxiety disorder NOS. Maternal and infant characteristics at both assessment points and stratified for both groups are presented in n = 2 mothers the interactive situation was missing at the 5-year follow-up. Consequently, only N = 30 dyads were analyzed for dyadic interactive abilities.At the 5-year follow-up, only a sub-sample of Assessments took place at the Heidelberg University Hospital, Germany. The study was approved by the independent ethics committee of the Heidelberg University Medical Faculty. According to the Declaration of Helsinki, written informed consent was obtained from all mothers both for their own participation as well as for the participation of their infant prior to the first assessment and after a full explanation of all study procedures. At first assessment, the Face-to-Face Still-Face paradigm FFSF;  was vide1), immediately after (C2) and 20 min after the FFSF paradigm (C3). Even though the circadian rhythm of the HPA-axis is not fully developed during the first months postpartum . However, n = 14 infants (22.2% of study sample) were assessed after 11 AM  due to postponed study appointments. Thus, we considered sampling daytime as a confounder. Furthermore, cortisol reactivity is correlated with prior napping and feeding. Consequently, additionally to the instruction of keeping the infants rested and fed as usually, time to and length of prior feeding and sleeping periods were considered as additional confounders. Salivary cortisol levels were interpreted on the basis of the area under the curve with respect to increase (AUCI) which allows a sensitive measure of physiological changes over time  were in range of normative parameters , indicating that cortisol levels declined from the first (C1) to the last (C3) assessment. In the group of infants with three valid cortisol measurements (n = 42) there were n = 13 responders (31%); responders were defined as infants whose AUCI was one SE (2.63 ng/ml × min) above zero. Low to medium responder rates and consequently declining cortisol means are often found in infant and child samples  were identified for the AUCI measure. Furthermore, there were no significant correlations  with potential confounder variables . Consequently, we disregarded these factors as confounders for the main and additional analyses.For two infants the Crameters . Mean of samples . HoweverThe German version of the Structured Clinical Interview for DSM-IV Axis I Disorders SCID-I,  is semi-st assessment and the 5-year follow-up were assessed with the German version of the Agoraphobic Cognitions Questionnaire (ACQ), the Body Sensations Questionnaire (BSQ) and the Mobility Inventory (MI)  . The thrst assessment were as follows: ACQ: M = 1.33 , MIA: M = 1.31 , MIB: M = 1.11 , BSQ: M = 1.69 . Anxiety questionnaire data was missing for n = 3 women in the 5-year follow-up sample, thus the sample size for analyses controlling for current anxiety symptoms was reduced. The mean scores for the anxiety questionnaires at the 5-year follow-up were as follows: ACQ: M = 1.31 , MIA: M = 1.30 , MIB: M = 1.13 , BSQ: M = 1.68 . In this study, we used a composite score averaged over the four scales for each assessment to encounter symptom heterogeneity between the different anxiety disorders. This composite score reached an excellent internal consistency  and ranged from min = 1.02 to max = 2.51  at the 5-year follow-up respectively from min = 1.00 to max = 2.36  at the 1st assessment.The mean scores for the anxiety questionnaires at the 1M = 31.92  for the first, M = 30.41  for the second and M = 31.63  for the third trimester. The intercorrelations between pregnancy trimesters were as follows: the first trimester significantly correlated with the second  and third trimester  as well as the second trimester correlated significantly with the third trimester . A composite score averaged over the three trimesters was used for the analyses This composite score reached an excellent internal consistency (α = 0.96) and ranged from min = 2.42 to max = 86.97 .The Prenatal Emotional Stress Index (PESI) is a self-report questionnaire assessing emotional stress during pregnancy . It was th revision of the macro-analytical tool “Coding Interactive Behavior” (CIB) (At the 5-year follow-up mother–child interaction during a free-play situation was coded by a trained and reliable coder (inter-rater-consistency = 87.6%) using the 4r” (CIB) . The codThe CIB is a valid, reliable and widely used  global rM = 3.67  for reciprocity and M = 1.70  for dyadic negative states.In this study, we focused on both dyadic composites, i.e., “reciprocity”  and “dyadic negative states” . The means were Statistical Package for Social Sciences (IBMTM SPSS® v. 24.0.0.0). G-Power v. 3.1.9.2  were valid for our sample and analyses by the use of Little’s MCAR-test . If the U-tests were carried out to compare the study groups regarding emotional stress, infant cortisol reactivity as well as CIB scores as the distributions of questionnaire data, cortisol values and reactivity as well as dyadic negative states significantly deviated from normal distribution (p < 0.01 in Kolmogorov–Smirnov and Shapiro–Wilk test). Especially for small samples and unequally sized groups, general linear modeling may not be sufficiently robust against violations to mathematical assumptions . Thus it may lead to progressive statistical testing . Consequently, repeated measures dfs were Huynh–Feldt corrected.Effects of group, measurement time and their interaction term on infant cortisol measures were tested by a two-way ANOVA for repeated measures with the between-subject factor “group” and the within-subject factor “measurement time” to account for differences in the infants’ cortisol trajectories between the groups and for intraindividual variance. Since mathematically premises are violated (see above) the results of this ANOVA are of purely descriptive quality. Mauchly’s procedure was used to test for violation of the assumption of sphericity. This assumption was violated for infant cortisol values  was used to control for and to evaluate the independent contribution of study variables  in explaining the CIB scores.We used a two-tailed critical α-error of α = 0.05. The valid sample size varies as a function of missing values in the original data and the 5-year-follow-up.2 = 1,071.80, df = 1,168, p = 0.98), indicating that the list-wise case-exclusions were valid for our sample and that this sub-population was representative for the total sample. Additionally, tests on comparability between the excluded and included cases revealed no systematic differences . Moreover, no differences were found between the groups regarding sociodemographic or birth-related data  for n = 42 infants (66.7% of study sample). However, the infants of the remaining sample (n = 21) had at least one valid cortisol value. Furthermore, n = 8 PESI-scores (12.7% of study sample) for each trimester as well as anxiety questionnaire scores at the first assessment  were missing. We estimated the missing values for these dyads using multiple imputations . Estimated cortisol values were restricted to the limit of detection of the cortisol assay (0.1–15.0 ng/ml), PESI and anxiety questionnaire scores were restricted to the maximum score range . I, the PESI and the anxiety symptoms composite were then additionally computed for every case including cases with imputed values in every imputed data set.We had complete cortisol data  was not due to just missing values but real study dropouts over an extended period and since the percentage of missing data exceeded 50%, we refrained from estimating missing values for the 5-year follow-up. All analyses were carried out for (1) the original data set and (2) in each of the 20 imputed data sets. The results of (2) were pooled. Consequently, two results are reported: the pooled results over the imputed data set and the results of the original data set.Since the missing interaction data of the 5-year follow-up in U-tests on comparisons between the clinical and the control group. Only emotional stress during pregnancy  differed between the groups in the pooled (p < 0.01) and the original data set (p < 0.01). The clinical group reported higher scores of emotional stress compared to controls . There were no group differences between the clinical and the control group regarding infant cortisol reactivity  or CIB scores .st assessment  and the 5-year follow up  was positive and significant. The intercorrelation between anxiety symptoms at the 1st assessment and the 5-year follow-up was positive and significant in the pooled  and the original data set .The intercorrelation between emotional stress and anxiety symptoms at the 1I), we used a generalized linear backward regression controlling for anxiety symptoms at the 1st assessment. As maternal disorder was not revealed as meaningful for infant cortisol reactivity, it was neglected in this analysis. In the first step, anxiety symptoms at the 1st assessment were excluded as non-significant for infant cortisol reactivity . The second and final step .To evaluate the unique and independent association between emotional stress during pregnancy  and infant cortisol reactivity . Multicollinearity seems unproblematic, as the VIF was low for both variables and both data  .U-tests, large effects (d = 0.8) in our pooled sample could be detected by a chance of 1-β = 0.69 . Consequently, especially for medium-sized and small effects the power of these comparisons is not sufficient. The power for the final regression model was approximated for the linear multiple regressions with one coefficient. Large effects (f2 = 0.35) for beta weights in our pooled sample could be detected by a chance of 1-β = 0.99 . The chance to find medium-sized effects (f2 = 0.15) in our pooled sample was 1-β = 0.86 . Small effects (f2 = 0.02) had a chance of 1-β = 0.20  to be detected. Thus, especially small effects could not be sufficiently detected.For the Mann–Whitney F = 1.83, p = 0.24; original data: F = 3.24, p = 0.06] nor of group . Moreover, the interaction effect between measurement time and group was non-significant . Means and standard deviations are demonstrated in There was neither a significant main effect of measurement time . The results of the ANOVA  with the transformed values as dependent variable remained unchanged: There is neither an effect of time , of group  nor an interaction effect .Although there is a widely accepted practice of log-transforming raw data to achieve normally distributed values , this tef = 0.40) between-subject effects in this ANOVA was high  and medium-sized (f = 0.25) within-subject and interaction effects were sufficiently detectable . Only medium-sized (f = 0.25) and small (f = 0.10) between-subject effects as well as small (f = 0.10) within-subject and interaction effects could not be excluded in these analyses . It can be concluded that for between-subject effects only large effects can be ruled out. For within-subject effects large and medium-sized effects can be ruled out.The power to detect large (I) and maternal anxiety symptoms at both assessments with dyadic negative states and dyadic reciprocity (CIB), we used stepwise backward regressions  with these predictors as main effects.As maternal disorder was not revealed as meaningful for dyadic interaction quality, it was neglected in these additional analyses. To evaluate unique and independent relations of emotional stress during pregnancy , infant cortisol reactivity  and anxiety symptoms at the 1st assessment . For dyadic reciprocity the same variables were excluded, however, in reversed order: Anxiety symptoms at the 1st assessment  and emotional stress . The third and final step  and maternal anxiety symptoms at the 5-year follow-up  were both associated to dyadic interaction quality.The following variables were stepwise excluded as non-significant for dyadic negative states: Emotional stress , we refrained from repeating these analyses with ln-transformed data despite skewed infant cortisol raw and reactivity values.As in these analyses generalized linear modeling was used to minimize effects of violations of mathematical assumptions  and as nMulticollinearity for these models was approximated by linear regression and the estimation of the VIF. For emotional stress , anxiety symptoms at the first assessment , at the 5-year follow-up  as well as for infant cortisol reactivity  multicollinearity seems unproblematic, as the VIF was low for all variables and both data .f2 = 0.35) for beta weights in our pooled sample could be detected by a chance of 1-β = 0.87 . The chance to find medium-sized effects (f2 = 0.15) in our pooled sample was 1-β = 0.53 . Small effects (f2 = 0.02) had a chance of 1-β = 0.12  to be detected. Thus, medium-sized and small effects could not be sufficiently detected in our 5-year-follow-up sample.The power for the final models was approximated for the linear multiple regressions with two coefficients. Large effects  retrospectively assesses emotional stress during pregnancy via self-report which may have affected postpartum answering tendencies. Moreover, it would be of great interest to assess saliva cortisol samples during pregnancy, enabling future studies to analyze the impact of maternal stress during pregnancy for child development prospectively. Fourthly, infant salivary cortisol was assessed prior to, immediately after and 20 min after the Still-Face paradigm. Due to few samples or the small time frame it is possible that we missed peak cortisol reactivity times, which may in part account for the negative mean cortisol reactivity. Lastly, the study design was observational, causality assumptions are not appropriate.Taken together, our empirical results as well as theoretical assumptions emphasize the importance to further investigate the influence of stress during pregnancy for infant and child development. Our results underline that emotional stress during pregnancy is linked to infant stress reactivity and this in turn influences mother–child interaction up to pre-school age. Regarding maternal stress and its influences on infant and child development, it would be of major importance to disentangle different time-effects as well as different kinds of stressors, such as psychological stress, anxiety, or depressive symptoms and life events . Furthermore, potential moderators should be addressed, such as early life experiences, stress coping strategies, social support or maternal face processing, since current studies show the detrimental effect of anxiety . With reThis study was carried out in accordance with the recommendations of the independent ethics committee of the Heidelberg University Medical Faculty with written informed consent from all subjects in accordance with the Declaration of Helsinki.A-LZ and MM contributed to the analysis and interpretation of the data, drafting of the manuscript, and final approval of the final version of the manuscript. NN and BD contributed to the study and manuscript conception, and approval of the final version of the manuscript. CR contributed to the study conception and design, drafting of the manuscript, and final approval of the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Given the promise of DMPA-SC to increase community-level access to modern contraception in developing countries, we conducted an observational study to assess the acceptability and feasibility of DMPA-SC self-injection among women in Kinshasa, Democratic Republic of the Congo, and of medical/nursing (M/N) students as instructors for self-injection.Women who selected DMPA-SC at a community outreach event adjacent to a health center were interviewed upon acceptance (baseline) and then 3, 6 and 12 months later.Of 850 clients selecting DMPA-SC at baseline, 640 (75.3%) opted for self-injection over being injected by the M/N students for reasons of convenience and personal agency. Among these 640 self-injectors, 47.5% were anxious at baseline (for fear of needles or injecting incorrectly). Over 80% reported feeling very ready after training, confident that they knew how to self-inject and confident that they would remember the next injection date. By 3 months, 97% described it as easy. Half (54%) experienced side effects, mainly menstrual irregularities, the main reason for discontinuation. At 6-month follow-up, self-injectors cited effectiveness and ease of use as positive elements, though one quarter reported side effects. Their impressions of M/N students as instructors were highly positive.Where DMPA-SC was free and easily accessible, the majority of women interested in DMPA-SC opted to learn self-injection. The M/N students performed well in instructing women to self-inject. Clients were highly satisfied with the services received, yet many did not recognize their student status, possibly because outreach occurred near a health facility. Once told, clients remained very favorable, suggesting strong motivation to receive their preferred contraceptive free, whoever the provider.This study provides additional evidence on the acceptability and the feasibility of the self-injection of DMPA-SC by users from a resource-limited setting. A 2015 pilot study in Kinshasa used medical and nursing (M/N) students to deliver DMPA-SC and other methods at the community level. It showed that women were highly satisfied with the method and service received from these providers 22.1Action en Santé et Dévéloppement (ASD), which has extensive experience in family planning training, to oversee the implementation of the intervention. Specifically, ASD arranged for participation of one medical and five nursing schools, including supervisors and students from the 2015 pilot; obtained relevant health zone authorization; developed training curriculum for educating/coaching women to self-inject; and coordinated free family planning “campaign days” in the community with health zone personnel. The students received a vest with a family planning logo and knapsack containing contraceptives, supplies  and data collection forms.We carried out the study in three of the 35 health zones in Kinshasa: Kintambo (urban), Lingwala (urban) and Nsele . The research team partnered with a local NGO, On campaign days previously announced to the community, 10–15 students arrived at a location adjacent to a health center and provided counseling and services  onsite to eligible clients. The students referred women to fixed facilities for methods requiring a trained family planning provider for initiation . At baseline, women interested in DMPA-SC were encouraged to try self-injection, but they could instead choose the provider-administered injection.mousse ; they then self-injected as the M/N student supervised. Students assessed competency to self-inject (on their own body) based on a checklist of 23 items that included Uniject preparation, skin disinfection, DMPA-SC injection and waste disposal, among others. Clients had to correctly perform 80% (> 18) of these items to be declared competent by the M/N students.Women opting for self-injection at baseline were invited to practice injection on a 2.2Data collection for this research consisted of (a) surveys conducted by trained interviewers at baseline and follow-up at 3, 6 and 12 months among DMPA-SC clients; (b) a survey among the M/N students about their experience as providers; and (c) in-depth interviews with MOH and health zone personnel. This analysis is limited to the acceptor surveys (baseline and follow-ups). Interviewers entered data on Android smartphones that have been programmed with the Open Data Kit application; data were immediately transferred to a server, which the research team regularly monitored.2.2.12.2.1.1The intervention and data collection took place simultaneously in the three different health zones. Female interviewers experienced in contraceptive surveys received refresher training on the questionnaire content and survey procedures; they were present on campaign days. After a woman accepted DMPA-SC — either via self-injection or injected by the student — she was invited to participate in the baseline study. Clients who accepted the interview then moved from the student to the interviewer, who obtained informed consent. We aimed to enroll all DMPA-SC acceptors. However, because some acceptors were unable to wait while interviewers completed other interviews, we reverted to a convenience sample of clients available for the interview after receiving DMPA-SC. At baseline, interviewers obtained contact information for all participants willing to participate in follow-up surveys.On all rounds of data collection, the wording on questions regarding the client's experience included four categories that the interviewer read: “very ___,” “somewhat___,” “not very ____” and “not at all” .2.2.1.2Community agents working for the health zone publicized a second round of campaign days, at which all DMPA-SC acceptors could receive a second dose. For clients who failed to return for the 3-month follow-up on campaign days, interviewers attempted to locate them in their homes or by phone. Because the research focused on DMPA-SC self-injection, only clients judged competent to self-inject by the 3-month follow-up were retained in the sample for the 6- and 12-month follow-up. At 3 months, these clients received three doses of DMPA-SC to cover home self-injection at 6, 9 and 12 months.2.2.1.3After the first two rounds, there were no further campaign days. The research team located self-injectors (1) by phone, to establish a time and place for the interview, or (2) in their homes, via the community extension worker. 2.3The researchers used Stata (version 13) to complete a descriptive analysis of the data.χ2 statistics for categorical variables and analysis of variance (ANOVA) for continuous variables to compare the baseline sample to each follow-up sample on sociodemographic characteristics. We assessed statistical differences at an alpha level of .05.To assess a possible selection bias resulting from attrition, we treated each survey  as independent from each other; then, we conducted bivariate analyses, Although this approach might not be best suited for longitudinal designs and could potentially lead to a lower likelihood to find significant differences, it was the best available approach given that we were unable to track participants as a unique longitudinal sample.This research received human subjects approval from Tulane University (#911338-7) and the Kinshasa School of Public Health (#ESP-CE/071/2016). All the participants provided written consent before their inclusion in this research.33.1On the first campaign days held for the purposes of this study, a total of 850 women chose DMPA-SC as their method. Of these, 640 women (75.3%) opted for self-injection over an injection by the M/N student. After being trained, the clients self-injected in front of the student. Women choosing not to self-inject cited fear of injecting incorrectly, fear of hurting themselves and greater trust in the provider (data not shown).3.2At baseline, the 640 DMPA-SC self-injectors were 27 years old on average; only one in five had completed secondary school. Three quarters were married or in union; close to 100% had at least one child, with the average being three; two thirds wished to have more children, often after several years. Only half were employed  .Table 1SThis study was conducted in a community context, without the benefit of client records available through fixed health facilities. We experienced severe loss to follow-up between surveys. Due to logistical problems, the student providers and interviewers returned 1 week late for the designated 3-month follow-up/campaign days . Interviewers were present on the designated campaign days for the rest of the 3-month follow-up . They interviewed all baseline respondents (provider-injected or self-injected DMPA acceptors) who attended, as well as attempting to locate others by phone or home visit.At 3-month follow-up, 131 cases (15.4%) were lost to follow-up. Among the 719 located, 504 (70.1%) self-injected at 3 months; 68 (9.5%) reported discontinuing DMPA-SC; eight (1.1%) had a pending injection; 130 (18.1%) were interviewed but dropped from the analysis because of the above-mentioned delay of resupply/interview at 3 months; and nine (1.3%) requested injection by the provider. At 6 months, an additional 129 (17.9% of 3-month sample) were lost to follow-up. Among the 375 clients located, 239 (63.7%) reported to have self-injected at 6 months, 40 (10.7%) had discontinued DMPA-SC, eight (2.1%) received the injection from someone else, and 88 (23.5%) still expected to self-inject. By 12 months, we located 204 clients; 136 (66.7%) reported to have self-injected at 12 months, whereas 24 (11.8%) had discontinued DMPA-SC, three (1.5%) received the injection from someone else, and 41 (20.1%) still expected to self-inject; see 3.3Less than half of the self-injectors (46.3%) had ever used a contraceptive method; 15.8% had ever used an injectable, and 2.2% had used DMPA-SC . Among pFor 61.7% of the self-injectors, DMPA-SC was their method of choice; almost all the remainder would have preferred an implant. Half (48.2%) came for services based on their own decision; for 37.8%, it was a joint decision with their husband or partner. Among those married or in union, about three quarters reported that their husband or partner was favorable to family planning. However, at least a quarter felt that some members of their community, including family and friends, opposed family planning (data not shown in tables).3.4n=640), most acceptors chose self-injection because it would be easy to perform at home (58.8%); one in five also mentioned liking to learn new things and to be able to manage this product themselves. Four in five considered themselves to be “very motivated” to try self-injection. Almost half (47.5%) reported being somewhat or very anxious at their first injection because of fear of the needle or pain. Close to 30% reported pain during the first injection, but less than 10% felt pain afterwards (data not shown.)At baseline (jeton) to remember the date of the next injection, with far fewer mentioning the calendar, a written note or “just remembering.” Only 8.0% expressed that they would want to seek help from a family member or someone else for the next injection.At baseline, 65.2% reported the first self-injection was somewhat or very easy (those finding it difficult mentioned problems of inserting the needle and pumping the medication). Most (90.3%) felt very well prepared by the training from the M/N students to perform self-injection. Over 90% felt confident to perform self-injection, to follow the instructions in the booklet on self-injection and to remember the date for their next injection . Most (6n=504), the experience with self-injection was similar, though 86.9% mentioned feeling less anxious and 90.5% found it less difficult than at baseline. Nine in 10 (89.1%) felt very confident that they had self-injected correctly. Half (54.1%) reported side effects, the most common being irregular period, heavy and frequent bleeding, no period and abdominal pain. Some 20.4% had experienced skin reactions at the site of injection after self-injection, but among these women, only 1.9% had sought treatment for the reaction (At 3 months (reaction .Table 4SConfidence remained high that they would remember the date of the next injection (98.7%) and that they would correctly perform it next time (99.5%).n=239), respondents cited the effectiveness of DMPA-SC in preventing pregnancies (66.5%) and its ease of use (45.2%) as the most positive aspects of DMPA-SC self-injection. The negative aspects of self-injecting DMPA-SC included its side effects (25.5%) and unavailability of the method (5.4%); however, 43.1% reported no negative aspects of self-injecting DMPA-SC (At 6-month follow-up ( DMPA-SC .n=136) produced few new results compared to the 6-month follow-up; 13.5% still found self-injection “as or more difficult” as when they started, with inserting the needle and expressing the medication still the main reasons. By contrast, 61.8% reported no negative aspects to self-injection.The findings from the 12-month follow-up  were their confidence that they could do self-injection and the convenience of not having to go to the health facility.3.5n=45), partner opposed (n=12) and change to another contraceptive method (n=11). At 6- and 12-month follow-up, discontinuers frequently cited fear of side effects (data not shown).Given the large loss to follow-up between rounds of data collection, we have not presented data on discontinuation rates. However, of 76 self-injectors who discontinued between baseline and 3 months, the main reasons were the desire to stop the method  reported that 3 months earlier, they had received at least one dose of DMPA-SC to take home . Almost all had a safe place to store it at home . Most disposed of the waste in trashcans (51.5%), latrines (42.3%) or “discarded outside” (11.6%) (data not shown.)3.7This study also assessed client satisfaction at baseline with the performance of the M/N students in teaching women to self-inject. Four in five (80.5%) of the initial self-injectors did not realize that the provider was a student. Yet most were very (78.8%) or somewhat (9.7%) comfortable about learning to self-inject from a student. Most felt the M/N students were comfortable in explaining the method and its side effects (94.4%), and how to self-inject (99.4%). Close to 90% of acceptors were very satisfied with the information and counseling they received; 95.9% of acceptors would “strongly recommend” or “somewhat recommend” DMPA-SC self-injection to others.At the 3-month follow-up, the findings were similar: 95.6% felt that the M/N students were very knowledgeable; 87.9% considered the explanations to be clear; and 93.5% found the M/N students to be “very respectful” toward them.3.81.Is it possible for the D6 to train M/N students in family planning as part of their nursing curriculum?2.Once trained, are M/N students capable of instructing clients to self-inject DMPA-SC at the community level?3.Is the use of nursing students to deliver contraception in the community compatible with the existing system for delivering primary health care at the health zone level?“Feasibility” was not measured by a specific set of variables in a survey but rather by demonstration of the following:The experience of this research pilot provided strong evidence that all three are possible, further strengthening support for institutionalizing the family planning curriculum in the country's nursing schools.4This study demonstrated the feasibility of training M/N students to teach women at the community level to self-inject DMPA-SC in Kinshasa, DRC. It also showed that three in four clients interested in DMPA-SC were willing to try self-injection, at least in a setting where contraception was free and easily accessible.Clients reported high levels of satisfaction both with DMPA-SC as a method and with self-injection as a procedure. Despite initial anxiety, by 3 months, they gained a high level of confidence in their ability to self-inject; few reported difficulties with the procedure or pain from it. Also, clients gave positive feedback regarding the performance of the M/N students: they were knowledgeable about DMPA-SC, they gave clear explanations, and they were respectful of the clients.Our findings on the acceptability of DMPA-SC as a method are consistent with the results of the pilot introduction of the method in Burkina Faso, Niger, Senegal and Uganda from 2014 to 2016 An important limitation of the research was the high loss to follow-up of DMPA-SC acceptors interviewed at baseline, resulting from (1) the team's delayed return for the 3-month follow-up  and (2) difficulty in re-locating clients in communities where street names are not posted and houses often than have no numbers. Although clients could refuse the follow-up interview (when asked at baseline), some may have given false phone numbers to avoid further contact, especially if members of their household did not know they were using contraception.This observational study was conducted under real-life conditions in highly impoverished neighborhoods of Kinshasa, with the aim of approximating the likely conditions in future expansion of this approach. In contrast to other self-injection studies where clients were recruited and taught to self-inject in a clinical setting The implications of this high loss to follow-up over the course of the study are unclear. Data in Although not reported herein, results from in-depth interviews with MOH officials and health zone officials indicated widespread acceptance for DMPA-SC self-injection as a method and for the use of M/N students to motivate and instruct women in its use in the community. The research team disseminated results from the studies of acceptors, M/N students and health authorities in Kinshasa in November 2017 as a first step to authorization for scale-up of this approach. In September 2018, the Minister of Health endorsed the scale-up of this approach, in addition to two other community-level strategies 6ème Direction of the MOH, which is responsible for the network of nursing schools throughout the DRC.Efforts are already under way to institutionalize the use of M/N students for community-based distribution of contraceptives, including both DMPA-SC and Implanon NXT through the administer contraception, the scale-up of self-injection presents additional challenges, including the source of resupply and correct disposal of the device. DMPA-SC is still relatively scarce in Kinshasa  Whereas the pilot research provides a clear model for operationalizing the use of nursing students to


We propose that bias-modulated graphene-nanocrystallites (GNs) grown vertically can enhance the photoelectric property of carbon film coated on n-Si substrate. In this work, GN-embedded carbon (GNEC) films were deposited by the electron cyclotron resonance (ECR) sputtering technique. Under a reverse diode bias which lifts the Dirac point of GNs to a higher value, the GNEC film/n-Si device achieved a high photocurrent responsivity of 0.35 A/W. The bias-modulated position of the Dirac point resulted in a tunable ON/OFF ratio and a variable spectral response peak. Moreover, due to the standing structured GNs keeping the transport channels, a response time of 2.2 μs was achieved. This work sheds light on the bias-control wavelength-sensitive photodetector applications. Moreover, the transfer technique for the graphene/silicon heterojunction is complicated and limits the material’s industrial applications. Therefore, a method to mass-produce a photodiode with low cost and high responsivity is highly desired.Photoelectric sensors are widely used in optical communication, infrared ranging, environmental monitoring, military security inspection, biomedical imaging, and scientific research ,2,3,4. G2/Si heterojunctions. The device size of the a-C film photodetector can be as large as a commercial photodetector (~tens of mm2). However, the responsivity of a-C film is still rather low.Amorphous carbon (a-C) thin films were synthesized from coal as a solid carbon source . The a-CRecently, we proposed a method for the direct preparation of graphene nanocrystallite-embedded carbon (GNEC) film via a low energy electron irradiation technique . Unlike EF) of GNs and help to form a p–n junction with n-Si. The photoelectric properties of the GNEC film/n-Si heterojunction were tested, and the mechanism of the nanostructure’s effect on the photoelectric properties of the GNEC coating was unveiled.In this paper, we report the photo-electric behavior of GNEC films coated on n-Si substrates prepared by electron cyclotron resonance (ECR) plasma sputtering technology under bias voltage modulation. The external bias could modulate the Fermi level (−2 Pa. The mirror confinement magnetic field was applied in order to enhance the plasma density. A negatively biased glassy carbon target is known to attract Ar+ ions to sputter carbon species towards the substrate [Vdep) was applied on the substrate to attract electrons [Vdep = 20, 50, and 80 V. The nanostructures of the carbon films were analyzed using Raman spectroscopy  and transmission electron microscopy .The GNEC films were coated on n-Si using an ECR plasma sputtering system. A detailed description of the sputtering system was reported in our previous works. A 500-W microwave was delivered into the vacuum chamber to generate the plasma at an argon pressure of 4 × 10ubstrate . The sublectrons . With th2. A Keithley 4200-scs semiconductor characterization analyzer was used to measure the I–V curves. To measure the frequency response, a diode laser  was modulated ON/OFF with a 50% duty cycle by a 100-kHz square waveform generated by a function generator . The rise/fall time of the modulated diode laser was 10 ns. The spectral response was measured by a white light source, a monochromator, a chopper, and a lock-in amplifier. A xenon light in the visible to near-infrared (300 nm to 1100 nm) range was used as the white light source. The xenon lamp light was dispersed by a monochromator (precision of 0.5 nm) and the average power was in the order of 10 μW/nm.A finger-type 50-nm-thick gold electrode was deposited on top of the GNEC film using UV lithography . A 50-nm2 square pieces. A finger-shaped electrode was deposited on the surface of the GNEC film by UV lithography, and a 50-nm flat gold film was deposited on the back of the Si substrate. The finger-type mask was designed with a central spine (~300 μm width) and two sides of branches (~150 μm width). The spacing between the two branches was ~500 μm. The spine and branches were all designed in the shape of wedges to collect the electrons efficiently. High-resolution transmission electron microscopy (TEM) was used to explore the cross-section sample of the 50-V film, as shown in The carbon films were coated on the n-Si substrate using an ECR sputtering system, as shown in Vdep = 20 V, 50 V, and 80 V are shown in (0001) = 5.98/2 nm−1 = 2.99 nm−1. Thus, the interplanar distance can be calculated as d = 1/2.99 nm = 0.334 nm, in accordance with the (0001) facet distance of multilayer graphene. In the green square area, the FFT image does not show a pattern, indicating no GN formation. In the TEM image, we used white circles to indicate the boundaries of the GN on the GNEC film. The average size of the white circles indicates the width of the GN boundary. At Vdep = 20 V, the carbon film has an a-C structure. At Vdep = 50 V, the average size of the GN boundary is ~4 nm. At Vdep = 80 V, the average size of the GN boundary is increased to ~15 nm.Transmission electron microscopy (TEM) and Raman spectra of carbon films coated under ECR low-energy electron irradiation at Vdep = 20 V, 50 V, and 80 V are shown in Vdep = 20 V, the D, G, and 2D profiles are vague and wide, and the intensity ratio between D and G peaks (ID/IG) is almost 1, indicating the formation of a-C structures. At Vdep = 50 V, a stronger 2D peak around 2700 cm−1 appears and the ID/IG increases to 1.34, indicating the formation of small-size GNs in the carbon film. At Vdep = 80 V, a sharp 2D peak can be observed and ID/IG increases to 2.25. This indicates that, with the increase of low-energy electron radiation energy, the structure of GN on GNEC film tends to be ordered and the width of the GN boundary tends to increase.Raman spectra of films at depV. ω decreases from 2693 to 2688 cm−1 and then to 2683 cm−1 as depV increases from 20 to 50 V and then to 80 V. The shift of the 2D band can be explained by the edge quantum effect theory [K/μE)1/2/(kKd), among the shift of the 2D peak (Δω) from the ω(Bulk) of large graphene bulk to the nanocrystallite ω(K), the size K, the average bond length (Kd), the average bond energy (KE), and the reduced mass of the dimer atoms (μ). Thus, as the crystallite size increases, the average bond length expands and the average bond energy drops, leading to the decrease of ω towards the reference point of large graphene. Thus, the trend of the red shift of ω(K) as depV increases agrees well with the theoretical prediction, verifying the increase of the nanocrystallite size of GNEC film.t theory ,36. ThisVdep = 20 V had an amorphous structure, a low photocurrent was generated under reverse bias. At a diode bias of −5 V, the GNEC film at Vdep = 50 V produced a larger photocurrent of 9 × 10−3 A. As the GN size increased, the GNEC film at Vdep = 80 V produced the highest photocurrent of 1.44 × 10−2 A at reverse Vdiode = −5 V. Increasing the irradiation voltage (Vdep) enhanced the crystallization of the GNEC film, leading to a larger photocurrent under reverse bias. Photocurrents are extremely low at zero bias and increase drastically with reverse bias. Graphene/n-Si [Vdiode = 0 V due to the Dirac cone of graphene. This behavior at zero bias is largely different from that of the GNEC/p-Si device [Vdiode = 0 V due to the natural p–n junction formation between the electron-trapped GNs and p-Si. In contrast, in GNEC/n-Si, the reverse bias could depress the GNs’ Fermi level to open up available energy states for holes to inject. Under illumination, the flux of photocurrent was limited by the density of states of graphene nanocrystallites near the Fermi level. Thus, the minimum photocurrent points in dep increased from 20 to 50 V and finally to 80 V  of GN, inducing the p-type behavior of GN and forming a larger junction with n-Si.V curves in darkness and at a power (P) of 10 or 40 mW at a wavelength of 785 nm laser are shown in ene/n-Si  also exhi device . The GNE80 V see . This isdiode were obtained. The GNEC/n-Si showed a broadband photo-detection ability and the peak position was located at the near-infrared region. The 80 V GNEC film showed a higher responsivity of 0.35 A/W compared to that of the 50 V GNEC film (0.27 A/W) under a 900 nm laser, due to the increasing crystallization of GNs in the 80 V film. Since the illumination power of the white light source was rather weak (~10 μW/nm), the R difference between −1 and −2 V was not as large as that in As shown in Generally, the increase in light responsivity is accompanied by an increase in the light response time . Unlike 2 and a complicated transfer technique is needed. In contrast, the a-C film device area is usually tens of mm2 and no transfer is needed for industrial applications. We aim to enhance the photoresponsivity of such devices by introducing standing structured GNs into the film. Despite its large area of 5 × 5 mm2, the performance of our device was outstanding compared with some other small-area plane-graphene devices. In comparison with the previously reported graphene photodetectors, we summarize some features of the previously reported photodetectors in In contrast to plane-graphene, GNEC film is a directly deposited carbon film which contains a large amount of standing structured GNs. Although plane-graphene has massless Dirac Fermions and presents an ultrafast response , the devEf (GN)) and n-Si (Ef (Si)) at thermal equilibrium in darkness. The Dirac cone shape on the left is an illustration of the band of GNs. In the film, it may not necessarily be that shape. The Dirac point represents the minimum density of states point. The edge states of GNs tend to capture electrons and raise the Ef (GN) [EV) and the bottom of the conduction band (EC) of n-Si. The Ef (Si) of n-Si locates near the valence band (EV) and bends at the interface with a potential barrier qVd due to the formation of a p–n junction. As shown in Ef ‘(Si)) and electrons of p-GN (Ef ‘(GN))—are formed in the non-equilibrium situation. An applied reverse bias raises the Dirac point of GNs, increasing the chemical potential disparity between GNs and n-Si and leading to the formation of junctions [qVr, preventing the dark current. The applied reverse bias can raise the Dirac point of GNs to a higher value, opening up a large number of accessible states for the holes to inject into and allowing a complete collection of the injected holes. Under illumination, photo-excited holes tend to flow into the accessible GN states. Hence, the electron-hole recombination rate is drastically reduced by GN trapping centers, leading to an outstanding responsivity.Under the irradiation of low-energy electrons, a large amount of standing structured GNs are vertically grown to provide a high density of edges for the GNEC film.  Ef (GN) . The twounctions . The potThere is huge potential for GNEC film to further enhance its responsivity, for example, by increasing the density of GN edges, preventing dark current leakage by introducing an interface insulator , nitrogeGNs vertically grown in a GNEC film can prominently improve the photoelectric performance of the material by improving its ability to collect photogenerated electrons and prolonging the lifetime of photoexcited carriers. The GNEC film/n-Si heterojunction exhibited a high photocurrent responsivity of 0.35 A/W under reverse bias, compared with the responsivity of 0.18 A/W of a-C film/n-Si. In the GNEC film preparation process, we can control the crystallinity by adjusting the energy of low-energy electron irradiation, so that the photoelectric performance of the GNEC film/n-Si photodetector can be improved.rise = 2.2 μs) under reverse bias. The excellent photoelectric performance exhibited under reverse bias was attributed to the GN crystallization and the increased Dirac cone. The bias-modulated position of the Dirac point resulted in a tunable ON/OFF ratio and a variable spectral response peak (from 900 to 1035 nm), enabling bias-control wavelength-sensitive photodetector applications. The GNEC film/n-Si heterojunction photodetector allows low cost, large area, and efficient production. It can be used for a variety of tunable photoelectric devices with high responsivities over broadband, having a broad application potential in industrial production.The GNEC film coated on n-Si exhibited excellent photocurrent responsivity (0.35 A/W) and a highly sensitive response time (τ